Methods for modulating metabolic pathways of micro-organisms and micro-organisms obtainable by said methods

ABSTRACT

The invention provides methods for changing the metabolic pathways of micro-organisms in the presence of a certain carbon source and uses of such changes, as well as micro-organisms and uses of such changes, as well as micro-organisms produced by these methods. In a preferred embodiment the invention provides new yeast strains with improved biomass yields, a process to obtain these yeasts and the potential application of these yeasts are provided. The biomass yield is improved by the introduction into a yeast of a DNA construct conferring altered expression of a gene encoding a protein controlling transcription of a number of glucose-repressed genes. The yeasts are less sensitive to glucose repression, resulting in an increase in respiratory capacity, reduction of ethanol production and increased conversion of sugar into biomass.

This application is the U.S. National Phase entry of PCT/NL97/00688 filed Dec. 12, 1997 and has benefit of priority from that filing date.

The present invention relates to the field of biotechnology, in particular to the field of culturing micro-organisms, in particular yeast.

Culturing of micro-organisms is a relatively old technique which is well established and well understood by persons skilled in this art. It usually involves bringing a micro-organism of interest into a culture medium wherein it can survive, grow and divide. The culture medium usually comprises all the necessary nutrients for the micro-organism to be able to do this.

Micro-organisms are cultured for many different purposes. These include the production of biomass, the production of antibiotics, the production of useful proteins expressed by micro-organisms (be it naturally or through genetic engineering), the production of micro-organisms useful themselves (for instance in brewing or baking bread, leavening of dough, etc.) Because of its relatively long history and its many uses the techniques for culturing micro-organisms have been very well optimised, so that further gains in yield or growth rate of the micro-organism to be cultured are difficult to achieve. However, because of the cost of culturing micro-organisms and the large amounts needed, such improvements (however small percentage-wise) remain very desirable.

One of the problems of culturing micro-organisms is that they often show preference for certain carbon sources, which carbon sources do not result in the best yields and/or growth rates of the micro-organism. Often the availability of such a preferred carbon source will lead to repression of the metabolism of other available carbon sources (which other carbon sources often do result in higher yields). For instance, S. cerevisiae, as many other micro-organisms, shows marked preferences for certain sources of carbon, nitrogen and energy. One such preference concerns the use of glucose above other fermentable and non-fermentable carbon compounds (see 1,2). This behaviour causes diauxic growth of this yeast when cultured on mixtures of carbon sources that include glucose. Yeast cells growing on glucose display high growth rates, presumably related to the ease with which intermediates can be derived from the catabolism of this sugar. Glucose has radical consequences for the enzyme complement and metabolic patterns in the yeast cell (FIG. 1). During growth on this sugar, enzymes required for metabolism of other carbon sources are either absent or strongly reduced in amount as a result of active degradation of mRNAs or proteins (catabolite inactivation), repressed synthesis of mRNAs (catabolite repression), or both. Such enzymes include permeases and key enzymes involved in the utilization of various sugars, enzymes of gluconeogenesis and the glyoxylate cycle. In addition, synthesis of components of the mitochondrial respiratory chain is repressed, resulting in a low respiratory capacity. Glucose-repressed cells, or cells pulsed briefly with excess glucose produce ethanol by decarboxylation of cytosolic pyruvate and subsequent action of alcohol dehydrogenase. This series of reactions, known as the Crabtree-effect, regenerates cytosolic NAD⁺ required for glycolysis. Although the basis of the Crabtree response is largely unknown (3), the occurrence of these reactions during large scale production of S. cerevisiae is undesirable because it reduces cell yield.

Suppression of ethanol production by yeast cells growing on glucose is currently achieved by limitation of the supply rate of the sugar. This procedure is only partially successful in that incomplete mixing can trigger a short-term Crabtree response. Additionally it suffers from the drawback that cells are forced to grow below their maximum capacity.

These or similar problems occur in many micro-organisms, particularly in eukaryotes, more in particular in yeasts. The present invention provides a general mechanism by which such problems may be solved in that it provides a method for producing a micro-organism of which a metabolic pathway has been modulated, for instance a micro-organism of which the sugar/glucose metabolism has been shifted from aerobic fermentation towards oxidation, comprising providing said micro-organism with the capability of inhibition or circumvention of the repression of the oxidative metabolism of glucose induced by the availability for the micro-organism of said carbon source. Surprisingly we have found that by simply interfering at a single (well-chosen) spot in the complex regulatory mechanisms of metabolic routes in (in particular) eukaryotic micro-organisms, it is possible to redirect said metabolic routes from one mechanism (fermentation) to another (oxidation). Because of the complexity of the regulatory systems surrounding metabolism it is generally believed that interference at a single point would be unlikely to be of any significance (because of all kinds of positive and negative homeostatic mechanisms which would restore the original situation) or would be deleterious if not disastrous (if it were capable of disrupting the feedback-mechanisms). We have found that a simple modification, in particular at the level of transcription activation does lead to the desired switch in metabolic mechanism, without disrupting the metabolism of the micro-organism.

In particular, the invention solves this problem when the preferred carbon source of the micro-organism is glucose, which is the case for many micro-organisms, in particular yeasts. One of the most important yeasts in industry is (of course) Saccharomyces cerevisiae. For that reason we have chosen this micro-organism as a model for explaining our invention. Because of its importance it is of course also a highly preferred embodiment of the present invention. Other well known industrial yeast strains such as Hansenula, Kluyveromyces and other strains are of course also within the scope of the present invention.

It will be understood that the result of the preference for glucose leads the metabolism down the aerobic fermentative pathway in many cases, as will be explained below. It will also be clear that for yield in biomass and/or production of useful proteins, etc. the oxidative/gluconeogenesis pathway is to be preferred. This pathway is often part of the metabolism that is repressed when glucose is available and which is used when other carbon sources are available. Thus an important aspect of the present invention is to provide a method according whereby the repressed metabolism is restored to a significant extent by activation of the pathways for metabolism for the non-preferred carbon sources.

It is preferred that said activation is achieved by providing the micro-organism with at least one transcriptional activator for at least one gene encoding an enzyme in said pathways.

A very suitable and preferred way of achieving said activation is one whereby the transcriptional activator is provided by introduction into the micro-organism of a recombinant nucleic acid encoding said activator. Said recombinant nucleic acid is preferably an expression vector. Such a vector may be an autonomously replicating vector, but it is preferred to use vectors that integrate in the host genome. However, it may also be achieved by other means, such as mutation (site directed).

There are two ways of having the transcriptional activator expressed. In one embodiment of the invention the transcriptional activator is constitutively expressed by said micro-organism. In an alternative embodiment the transcriptional activator can be expressed by the micro-organism upon induction, for instance by the presence of glucose. The person skilled in the art will be able to determine which one is to be used for different circumstances and desired end results. In many instances it will be preferred that the vector used to introduce the activator is capable of integration into the genome of the host. In other embodiments a self-replicating vector may be used.

A very efficient way of achieving the derepression of the metabolic pathways other than those active in the presence of glucose is one whereby the transcriptional activator provided is a Hap4 protein or a functional equivalent, derivative or fragment thereof. A functional equivalent or derivative or fragment is defined as a molecule still having the same activity in transactivating the relevant genes from the relevant pathways (in kind, but not necessarily amount). Apart from being useful for biomass production and other uses of the micro-organisms themselves, another important use includes the production of recombinant proteins, homologous or heterologous. Thus the invention also provides methods for producing micro-organisms according to the invention, which micro-organisms further comprise a nucleic acid encoding such a protein of interest. Many proteins of interest have been disclosed and have been produced in yeast or other micro-organisms. They do not need to be reiterated here.

The micro-organisms produced by the methods according to the invention are also part of the invention. They are improved in many aspects, when compared with the organisms they are derived from, for instance they can have improved biomass yield upon culturing; they may show increased glucose oxidation; they may display increased oxidative sugar metabolism and/or reduced aerobic fermentation. Normally, under anaerobic culturing conditions the micro-organisms according to the invention will behave essentially the same as the corresponding micro-organism not provided with the modulation of this metabolic pathways.

The process to obtain the improved micro-organisms, in particular improved yeast has applications in all industrial processes in which optimal conversion of sugar into biomass is required. Use of these improved yeasts will lead to reduction of costs because of reduced process times and increased amounts of biomass per consumed glucose. This invention is well applicable in, for example, the (aerobic) production process of yeast for bakeries, or as source of flavour-enhancing yeast extracts. Furthermore this invention will lead to increased production of metabolites and heterologous gene products, such enzymes, precursors for chemicals, biosurfactants and fatty acids for application in pharmaceutical, agricultural or food sectors.

Concerning the application in baker's yeast or brewer's yeast, it should be noted that the reduction of alcohol formation in the production phase does not negatively affect growth or alcohol production during anaerobic fermentation, which is crucial for leavening of the dough or for the brewing process. This invention reduces alcohol production only when the yeast can make use of its enhanced respiratory capacity, i.e. under aerobic conditions.

The invention may also be applied with respect to manipulation of glucose repression or even glucose inactivation of processes not directly related to respiratory function of yeast. The uptake and metabolism of carbon sources other than glucose, such as galactose, sucrose or maltose is repressed by glucose. Yeast fermentation in lean dough depends on maltose as main substrate, which is produced in the dough from starch by action of amylases present in the flour. The flour contains in addition variable amounts of other sugars amongst which glucose. Maltose-permease, responsible for the translocation of maltose across the plasma membrane and maltase, the maltose metabolising enzyme, are both subject to glucose repression and inactivation (4).

Since this negatively affects CO₂ production and thereby leavening activity, tools to reduce the glucose effect on maltose uptake and utilisation would be useful. Therefore it is noteworthy this invention will even improve leavening activity of said yeast strains.

DETAILED DESCRIPTION

As stated before, a very important micro-organism is Saccharomyces cerevisiae. We will explain in detail how its reaction to the presence of glucose may be changed, as exemplary for other micro-organisms. In the presence of glucose Saccharomyces shows a “Crabtree” response and switches to ethanol production through aerobic fermentation.

The present invention in one embodiment offers a solution to such problems by the construction of production strains of micro-organisms in particular yeasts, more specifically S. cerevisiae in which the Crabtree-effect is reduced or absent. The principle of the approach is the controlled de-regulation of glucose-repressible genes by overexpression of a specific transcriptional activator from a promoter insensitive to glucose control. The resultant shift in balance from fermentative to oxidative metabolism leads to increased growth rates and reduced ethanol production.

Glucose Control of Metabolism in S. cerevisiae

The extensive changes in enzyme complement during a shift from oxidative/gluconeogenic to fermentative growth are, in the vast majority of cases, the result of induction or repression of transcription of the corresponding genes in response to glucose. Genes whose expression is repressed by this sugar can be divided into three groups:

1. Genes required for the uptake and metabolism of other carbon sources, such as galactose, sucrose, maltose, glycerol, lactate and ethanol.

2. Genes unique to gluconeogenesis and the glyoxylate cycle.

3. Genes coding for Krebs cycle enzymes and components of the respiratory chain.

Although each group displays distinct features of regulation, a number of common transcription factors and mechanisms are involved and these form the main glucose repression/deprepression pathway (MGRP; 2). As shown in FIG. 3, key events in this pathway are the activation or inhibition of a number of key transcription factors in response to a signal generated by glucose. The nature of this signal is unkown. Its main effect is, however, to inhibit or counteract the action of the Snf1/Snf4 complex, a protein serine/threonine kinase, which is thought to alleviate transcriptional repression and promote transcriptional activation at glucose-regulated promoters (5). Although as yet, there is no evidence that Snf1/Snf4 directly phosphorylates a transcriptional regulator, genetic studies suggest that important direct or indirect targets for Snf1/Snf4 are the transcription factor Mig1, Ssn6/Tup1 and Hap2/3/4. Mig1 is a zinc-finger protein which acts as a transcriptional repressor at many glucose-repressible genes (6) Ssn6/Tup1 acts as a repressor of transcription at a large number of genes, probably in combination with gene- or family-specific transcription factors (7). The Hap2/3/4 complex is, in contrast, an activator of transcription. It is required for induction of transcription by non-fermentable carbon sources of a limited number of genes encoding proteins involved mainly in mitochondrial electron transport, Krebs cycle, haem biosynthesis and gluconeogenesis (8, 9, 10, 11, 12). Transcriptional regulation by the Hap2/3/4 complex is the main mechanism for coordinating the derepression of these enzymes in response to changes in carbon status of the medium (11, 13). The activity of the Hap complex is controlled by the availability of the activator subunit Hap4, whose synthesis is approximately 5-fold repressed by glucose (13).

Transcription and Transcriptional Control in Yeast

A typical yeast promoter consists of several cis-acting elements that function as target sites for regulatory proteins (FIG. 2). The position of transcription initiation by the RNA polymerase II complex is located at the initiation site (I). The TATA-box (T) has been shown to be essential in many promoters for transcription initiation to occur. It is the target site for the basal RNA polymerase II transcription factor TFIID, which nucleates the assembly of the other basal transcription factors and RNA polymerase II into a stable preinitiation complex. In addition to these basal control elements, at least one upstream activation site (UAS) is required for transcription. UAS elements function as DNA binding sites for transcriptional regulatory proteins, that are thought to interact with the basal transcriptional machinery to mediate specific regulation. In many instances, yeast promoters consist of several TATA- and UAS-elements, which together determine the rate of transcription of the adjoining gene. In addition, yeast promoters may contain operators or upstream repressor sites (URS) and upstream induction sites (UIS). By binding of specific proteins these elements contribute to the overall transcriptional regulation.

Carbon Source-dependent Transcriptional Regulation by the Hap2/3/4 Complex

Carbon source dependent transcription of genes encoding a number of components of the mitochondrial respiratory chain and enzymes of gluconeogenesis is regulated by the Hap2/3/4 complex. Hap2 and Hap3 were first identified as proteins capable of binding to the UAS2 region of the gene encoding iso-1-cytochrome c in S. cerevisiae (14). This region, responsible for carbon source response, contains a sequence motif closely resembling the CCAAT box element found in many other eukaryotic promoters. The two proteins bind to DNA in an interdependent manner. Hap4 appears not to contact DNA directly, but is necessary for DNA binding of the other two proteins. Sequence analysis reveals a C-terminally located, highly acidic region whose presence is necessary for activity. Replacement of this region by the activation domain of the yeast Gal4 protein restores activity, suggesting that it provides the principal activation domain of the DNA-bound Hap2/Hap3 complex (13). Transcription of the genes for Hap2 and Hap3 in S. cerevisiae is not substantially affected by carbon source, but expression of the gene for Hap4 is glucose repressible (13). This suggests that Hap4 is the key component of the complex in terms of its ability to regulate transcriptional activity in response to carbon source.

Insight into the structure of the Hap2 and Hap3 proteins has been obtained by the isolation and sequencing of the corresponding genes. The HAP2 gene encodes a 265 residue protein, of which an evolutionarily-conserved 65 amino acid core in the highly basic C-terminal region is necessary and sufficient for both complex formation and binding to DNA (FIG. 4). The HAP3 gene encodes a 144 residue protein, which contains a 90 amino acid core (B-domain) required for complex formation and DNA-binding (FIG. 5). The HAP4 gene encodes a 554 residue protein containing two highly acidic regions in its C-terminal domain (13; FIG. 6). Both of these appear to be necessary for transcriptional activation.

Genes corresponding to HAP2 and HAP3 have been isolated from a wide range of organisms (15). The encoded proteins form a heteromeric complex called NF-Y, CBF or CP, which activates transcription by binding to the evolutionarily conserved CCAAT box element. Hap2- and Hap3-homologous subunits make similar contacts with DNA. For the human CP1 complex, it has been shown that the Hap2 and Hap3 homologues are exchangeable in vitro with those of yeast (16). However, although the human CP1 complex consists of more than two subunits, none of these appear to correspond to the S. cerevisiae Hap4 protein. The Hap homologous complexes are not specifically involved in induction of genes under certain growth conditions, but function as general transcription factors required for basal level expression of a large number of genes.

As the Hap complex (in the yeast Saccharomyces cerevisiae) is involved in the regulation of many metabolic processes, it is to be expected that modification of its expression has a. profound effect on the cell's physiology. With regard to anabolic processes, to date not much is known about its role but with regard to the catabolic network (that is, the energy conserving machinery) in S. cerevisiae, synthesis of components of the respiratory chain (hence mitochondrial) is to a significant extent under control of the Hap2/3/4 complex. It seems justified to conclude that Hap-dependent regulation is at least involved in the physiological phenomenon known as the Crabtree effect. Its direct physiological impact is a catabolic shift from respiratory to fermentative catabolism whenever elevated levels of glucose are present. This results in a significant decrease in the efficiency of energy conserved: only 2 moles of ATP are synthesized per glucose fermented to ethanol and carbon dioxide, whereas the number of moles of ATP (equivalents) per glucose oxidized (to carbon dioxide) is manyfold higher (the exact number still being a matter of debate). An important indirect effect is a decrease in anabolic capacity: it is known that the maximal obtainable growth rate of S. cerevisiae is highest under conditions that allow respiration. As a consequence, whenever conditions are such that the catabolic flux into respiration is increased, the yield value (Y_(glucose), defined as the amount of cells obtained per glucose consumed) will be considerably higher and the organisms will grow faster. In other words, under such conditions the partitioning of the total carbon flux over the catabolic and the anabolic flux will be directed towards the latter. Thus, a larger part of the carbon source is directed towards biomass formation and a given biomass concentration is achieved in a reduced time span. Due to the said regulation (Crabtree effect), fully respiratory catabolism can only occur under aerobic conditions with continuously very low glucose availability, a condition not often met in practical settings.

According to the invention, the inhibition or circumvention of glucose-regulated partitioning of the catabolic fluxes should result both in increased Yglucose values and higher growth rates. Here, it is important to note that it can be foreseen that a relatively small increase in the respiratory flux may result in a significant gain in biomass yield due to the large difference in the energetic efficiency between respiration and fermentation. All anabolic processes will be enhanced and since anabolism comprises protein synthesis it is to be expected that said increase is beneficial not just to biomass production per se only but also to the production of specific proteins.

Modification of the expression of glucose repressed genes can be achieved by interference in other factors of the glucose repression signalling cascade besides Hap4. All mutations in upstream regulators of the cascade (see FIG. 3), like Snf1 or Hxk2, do alleviate glucose repression of SUC2, GAL, MAL and respiratory genes, but these mutants display a wide range of phenotypic defects (1, 17, 18) and are not suitable for industrial application of yeast. De-repression of SUC, MAL and GAL genes can also be accomplished by removal of the general glucose repressor Mig1, which results in partial alleviation of glucose control of maltose metabolism in a laboratory strain (19) This does however not affect glucose repression of respiratory genes, and no change in fermentative-oxidative metabolism, growth rate or cell mass yield.

We have now found that yeast, transformed with a construct that overexpresses the HAP4 gene, becomes insensitive to glucose repression of transcription of a number of genes, amongst which genes encoding respiratory components. This results in increased respiratory capacity of this yeast strain and reduced ethanol production. This altered aerobic sugar metabolism leads to a drastic increase in biomass yield and a significantly increased growth rate.

The present invention provides a transformed yeast with an reduced aerobic fermentation rate of glucose which comprises the introduction into yeast of a DNA construct which contains an homologous gene encoding a protein de-regulating glucose repression of a number of genes in the said yeast.

The homologous gene in this invention, HAP4 is cloned into a circular vector DNA construct which is transformed into yeast by a procedure described in the following section. The vector contains DNA sequences that enables replication in both E. coli and yeast, sequences that enable cloning of DNA fragments into the vector, a yeast marker gene and a bacterial marker gene that enables selective maintenance in yeast or E. coli respectively. After transformation of the vector into yeast, it will be self-replicating and be maintained as long as selective pressure against plasmid loss is sustained. During non-selective propagation (i.e. growth in the presence of leucine in this particular case), the plasmid will be lost. From a practical point of view, it is preferable to grow yeast non-selectively. This will entail alteration of expression of HAP4 by integration of the altered gene in the yeast chromosome. By “altered” is meant the exchange of the natural promoter by another promoter which is constitutively active, or by integration of a DNA construct consisting of such a promoter fused to the HAP4 gene on a different locus on the chromosome, e.g. the SIT2 locus (20). Integration of homologous yeast sequences is a well described and efficient technique (21) and can be easily applied to (industrial) yeast strains. The yeasts thus obtained are stable transformants and the altered HAP4 gene can be maintained in the genome without selective pressure.

Yeast with a chromosomally altered HAP4 gene can be constructed in such a way that the transformants are completely devoid of prokaryotic DNA, in contrast to the transformants also described in this invention which contain a plasmid harbouring prokaryotic DNA sequences. By integration of only homologous DNA, originating from Saccharomyces, one can improve the yeast without introducing any heterologous DNA.

The present invention comprises induction of changes in glucose metabolism by overexpression of a key transcriptional regulator of oxidative metabolism of yeast by introducing in the yeast a DNA construct expressing HAP4 under control of the ADH1 promoter, whose activity is enhanced approximately ten-fold by glucose. The elevated expression level has alternatively been achieved by using other strong promoters, which are constitutively expressed, independently of the presence of glucose. This is of particular importance for application in fed-batch cultures, where the glucose concentration is kept low in order to minimise the Crabtree-effect. Several promoters belonging to genes encoding enzymes of the glycolytic cycle, like glyceraldehyde-phoshate dehydrogenase (GPD) or genes involved in ribosomal expression, such as promoters for the transcription of elongation factors (EF) are well characterised and widely used for overexpression of yeast genes. These promoters have been isolated from S. cerevisae and cloned in expression vectors for yeast (22). The coding region of the isolated HAP4 gene was cloned behind these promoters, after which the promoter-HAP4 fusions were recloned in such a construct that the HAP4 gene with altered regulation of expression could be integrated into the yeast genome. This procedure leads to stable transformants which exhibit all the advantages described above due to more oxidative growth and which in addition do not contain any prokaryotic DNA sequences.

The yeast strains described in this invention comprise both laboratory strains and industrial production yeast strains. The overexpression of Hap4 was first achieved in a laboratory strain by means of introduction of a self-replicating plasmid harbouring the HAP4 gene under control of the ADH1 promoter. The metabolic behaviour of this engineered strain was tested in detail as described below, showing a significant increase in oxidative metabolism when grown on glucose. Industrial production strains were transformed with the same plasmid, which was slightly modified by introduction of a dominant marker thereby enabling selection of transformants containing the plasmid overproducing Hap4. The response of the industrial strains to Hap4 overexpression was similar to that of the laboratory strain, i.e. glucose repression of respiratory function is alleviated and therefore glucose metabolism has shifted from fermentative towards oxidative metabolism. To obtain stable yeast strains with all the advantages described above, genomic integration of constitutively expressed Hap4 was carried out, as described in detail in the following sections.

Experimental

Saccharomyces cerevisiae strain DL1 has been transformed with YCplac111::ADH1 (without any gene placed behind the ADH promoter) or YCplac111::ADH1-HAP4 (expression of HAP4 under control of ADH1 promoter). Transformation of the yeast strain with YCplac111::ADH1, the so-called “empty” plasmid is necessary to prevent any differences in physiology of the Hap4 overproducer and the wild type due to differences in e.g the plasmid encoded LEU2 marker gene. The nomenclature of the transformed yeast strains is as follows: DL1 denotes strain Dl1 transformed with YCplac111::ADH1, whereas DL1HAP denotes DL1 transformed with YCplac111::ADH1-HAP4. The expression level of HAP4 mRNA in these strains is depicted in FIG. 9. Expression of HAP4 in Dl1 is strongly repressed by glucose. Introduction of the plasmid with HAP4 under control of the ADH1 promoter leads to an increased expression level of HAP4 in DL1HAP which is grown on media containing 2% glucose. This level is comparable to the expression level of HAP4 in wildtype DL1 cells when grown on non-fermentable carbon-sources, which do not repress transcription of HAP4 and genes encoding respiratory components.

To study the effect of HAP4 overexpression on transcriptional control of respiratory function, we first studied the mRNA levels of different genes encoding components of the respiratory chain. As shown in FIG. 9, the elevated level of HAP4 in glucose containing medium leads to de-repression of transcription of QCR8, the gene encoding the 11 kDa subunit of the yeast ubiquinol-cytochrome c oxidoreductase (QCR) complex of the respiratory chain. Comparable results were obtained for a number of other genes encoding respiratory components (Table I). Transcription of SUC2, a glucose repressed gene without an Hap binding box in the promoter region is not induced by overexpression of HAP4 on glucose.

To test whether the increased level of mRNAs of respiratory components results in an higher respiratory capacity of the Hap4 overproducing strain, we measured oxygen consumption rates of cells as described in a previously section. Respiratory capacity of DL1HAP cells grown in shake flask cultures on complex media containing glucose is increased two-fold compared to wildtype cells (Table II). When grown in the presence of the non-fermentable carbon-source lactate, the respiratory capacity is further increased approximately five-fold to a level similar for both wildtype and Hap overproducing strains.

Further characterization of the physiological properties of the Hap4 overproducing strain required growth under controlled conditions (constant pH, aeration, stirring) in well defined mineral media (see section ‘batch cultivation in fermentors’). Therefore, aerobic growth of Dl1 in a defined mineral salts medium containing 30 g/l (3%) glucose was compared with growth of Dl1HAP cells. As calculated from the growth curves (not shown), DL1 grew exponentially with a specific growth rate of 0.16±0.01 h⁻¹, whereas the growth rate of DL1HAP was 0.18±0.01 h⁻¹. Overproduction of Hap4 thus results in an increased growth rate of 11%.

During a period of six hours during exponential growth, samples were taken with an interval of one hour to measure substrate consumption and biomass and product formation. The mean biomass yield during this time course, i.e. the amount of biomass formed per gram consumed glucose is 10.1 gram for DL1 and 14.8 gram for DL1HAP. Overproduction of Hap4 thus leads to 46% increase in biomass yield compared to wild type, which is rather constant during the experiment (see FIG. 10).

Analysis of other carbon compounds present in the culture medium showed that ethanol production is significantly decreased (38%) in DL1HAP compared to the wild type (Table IV). The reduction in ethanol formation is accompanied by a 2.3-fold increase of the amount of acetate, whereas the amount of glycerol decreased 3.5-fold in DL1HAP cells. The oxygen consumption during the experiment was approximately twice as high in DL1HAP compared to DL1. All data are thus consistent with a shift of carbon metabolism from fermentative towards oxidative metabolism due to overexpression of HAP4. This is further illustrated in Table V, which summarizes carbon fluxes in both strains. The amount of CO₂ produced via TCA cycle was calculated by the amount of oxygen consumed during the experiment.

When grown under anaerobic conditions, both strains are identical with respect to growth rate, ethanol production and biomass yield (data not shown). This implies that overexpression of HAP4 only exhibits its effect during aerobic growth of yeast. Processes depending on anaerobic alcoholic fermentation, like brewing or dough leavening will be unaffected by HAP4 overexpression. This invention is therefore very well applicable in optimization of biomass yields in the aerobic production phase of industrial yeast strains.

Taking into account strain-dependent variations in glucose repression (23, 19), it should be mentioned that the regulation of expression of HAP4 in several industrial strains was found to be similar to laboratory strains described above and in literature (13), i.e. the expression is repressed by glucose (data not shown). To obtain constitutive high expression levels of HAP4 in the industrial strains, we integrated a DNA construct consisting of a constitutively active promoter (of glyceraldehyde-phosphate dehydrogenase, GPD1 and elongation factor 1-alpha, TEF2) fused to the coding region of the HAP4 gene in the genome of the yeast strain. The integration plasmid is constructed in such a way that the promoter-HAP4 fusion is flanked by DNA sequences representing parts of the yeast SIT2 gene (see FIGS. 11 and 13). Integration of the total plasmid including the promoter-HAP4 fusion can therefore take place at the SIT2 locus in the yeast genome. The presence of a gene encoding acetamidase (amdS) enables cells which have integrated the plasmid to grow on medium containing acetamide as a nitrogen source, in contrast to untransformed cells which are unable to grow on this medium. Since the plasmid does not contain any sequences which enable replication in yeast, integration of the plasmid in the genome is required to acquire the ability to grow on acetamide.

Strains DS28911, DS18332 and DS19806 were transformed with the plasmid pKSPO2-GPDHAP4 or pKSPO2-TEFHAP4. Transformants with an integrated plasmid in the genome were selected on plates containing acetamide. Transformants containing pKSPO2-GPDHAP4 were named DS28911GH, DS18332GH,or DS19806GH, transformants containing pKSPO2-TEFHAP4 DS28911TH, DS18332TH or DS19806TH. In a number of transformants the position of integration was analysed by Southern blotting, in which genomic DNA fragments containing SIT2 and/or HAP4 sequences were visualized (see FIG. 13). The length of the different fragments revealed that integration preferentially took place at the HAP4 locus, as shown in FIGS. 13 and 14. These (anaploid) transformants contain both one or more glucose repressed HAP4 genes and a HAP4 gene which is under control of a constitutively active promoter.

To test the expression level of HAP4 in these transformants, mRNA was analysed of cells grown aerobically in media containing 2% glucose. The mRNA expression level of one selected transformant of every strain containing either the GPDHAP4 (GH) or the TEFHAP4 (TH) is shown in FIG. 15. Different levels of HAP4 mRNA were obtained in the different strains and with the two different promoters. The effect of overexpression of HAP4 on expression of QCR8, a glucose-repressed gene encoding a subunit of the respiratory chain was analysed in four independent transformants. Strains exhibiting clear and reproducible de-repression of QCR8 were selected for further studies and are shown in FIG. 15. As a loading control a constitutively expressed gene (PDA, encoding a subunit of pyruvate dehydrogenase, (24)) was visualized, showing that the higher expression of QCR8 is due to HAP4 overexpression. The integrated constitutively expressed HAP4 thus appears to result in alleviation of transcriptional repression of a respiratory component, analogous to the effect of the plasmid encoded HAP4 under control of the ADH1 promoter as described above.

To test the respiratory capacity of DS28911, DS18332 and DS19806 with the integrated GPDHAP4 (GH) or the TEFHAP4 (TH) fusion, we measured oxygen consumption rates of cells grown in medium containing 4% glucose. As shown in Table VI, overexpression of HAP4 on glucose results in an increase of he respiratory capacity, ranging from 2.2 to 4.1 fold the value found in the corresponding wild type cells. The respiratory capacity of cells grown in lactate containing medium is 7.7 fold higher than in wild type cells. The allevation of glucose repression of respiratory function is thus only partial, but similar or even higher than obtained with the laboratory strain DL1 containing the ADH1-HAP4 fusion on plasmid. It can therefore be expected that the physiological advantages of the latter strain are also applicable to the industrial strains harbouring the integrated constitutively expressed HAP4.

Application of the modified industrial strains requires removal of any sequences that are not derived from yeast. The transformants contain the total plasmid pKSPO2-GPDHAP4 or pKSPO2-TEFHAP4 in the genome. The ‘non-yeast’, plasmid sequences can however be removed by homologous recombination of identical DNA segments in the genome, as depicted in FIG. 13. Cells which are devoid of plasmid sequences can be selected by growth on medium containing fluoro-acetamide, which is toxic for cells still containing the gene encoding acetamidase. When the plasmid is integrated at the HAP4 locus, this recombination event will cause loss of the GPD- or TEF-HAP fusion as well, resulting in a wild type strain. Only after integration at the SIT2 locus the chromosomal arrangement is such that recombination can also result in cells that still contain the GPD- or TEF-HAP4 fusion. These ‘clean’ transformants now only contain yeast sequences and are suitable for industrial application.

To obtain these clean transformants, strains DS28911, DS18332 and DSl9806 were transformed with plasmid pKSPO2-GPDHAP4 or pKSPO2-TEFHAP4 which was linearized at the SfiI site (see FIG. 11B). This led to a higher efficiency of integration at the SIT2 locus, as shown by Southern blot analysis. Comparison of expected (FIG. 13) and obtained (FIG. 16A) DNA fragments of a number of transformants revealed a new set of strains overexpressing HAP4 from the SIT2 locus. The expression levels of HAP4, QCR8 and PDA1 were determined by northern blotting of mRNAs isolated from cells grown in medium containing 2% glucose. As shown in FIG. 17A, also in these strains the elevated expression of HAP4 resulted in alleviation of glucose repression of QCR8. Subsequently we selected fluoro-acetamide resistant cells that had lost their plasmid sequences (including the acetamidase gene) by homologous recombination. Transformants which retained the GPD- or TEF-HAP4 fusion in the genome were selected by verification of the correct chromosomal arrangement by Southern blot analysis. FIG. 16B shows three examples of clean transformants derived from transformants DS18332GH15 and DS18332TH25. As shown in FIG. 17B, removal of the plasmid sequences has no negative effect on either HAP4 overexpression or QCR8 derepression. The effect of the HAP4 overexpression in these strains on oxidative metabolism can therefore be expected to be similar to those described above.

EXPERIMENTAL PROCEDURES

Cloning Techniques

All general cloning techniques (plasmid isolation, restriction, gel electrophoresis, ligation) were carried out as described by Maniatis et. al., Molecular Cloning, A Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). DNA restriction enzymes were purchased from New England Biolabs (Biolabs), Boehringer Mannheim (Boehringer) or Bethesda Reseach laboratories (BRL). These enzymes were used in conditions and buffers described by the manufacturer.

Construction of Recombinant Plasmid YCplac111::ADH1-HAP4

The centromeric plasmids YCplac111::ADH1 and YCplac111::ADH1-HAP4 are capable of self-replicating in E. coli and in yeast and contains the ADH1 promoter region without (YCplac111::ADH1) or with the coding sequence of HAP4. The construction of YCplac111::ADH1-HAP4 is outlined in FIG. 7.

YCplac111::ADH1 is derived from YCplac111 (25) and contains between the BamHI and the SmaI site a 723 bp EcoRV promoter fragment from pBPH1 (26). Vector pBPH1 was derived from pAC1 (27), which is a YCp50 derivative which carries the BamHI/HindIII fragment from pMAC561 containing the yeast alcohol dehydrogenase I promoter (28).

The coding region of HAP4 was cloned behind the ADH1 promoter in the former construct by isolation of HAP4 from pSLF406 (13). pSLF406 was digested with BspHI, which cleaves HAP4 at position −1 relative to the start codon the coding sequence. The BspHI end was blunted and subsequently the HAP4 fragment obtained by cleavage of the DNA with PstI and isolation from agarose gel. YCPlac111 was cleaved with SmaI and PstI and ligated with the HAP4 fragment to generate YCplac111::ADH1-HAP4.

Construction of Recombinant Integration Plasmids p425-GPDHAP4, p425-TEFHAP4, pKSPO2-GPDHAP4 and pKSPO2-TEFHAP4

The plasmid pKSPO2 is used for integration of a fusion of HAP4 and the GPD1 or TEF2 promoter. These fusions were first constructed in shuttle vectors containing either the GPD1 (p425GPD) or the TEF2 promoter region (p425TEF) (22). A 2621 bp fragment from pSLF406 (13), containing the HAP4 gene, was isolated after digestion with BspHI, blunting and digestion with PstI as described in the previous paragraph. This HAP4 fragment was cloned in SmaI and PstI sites of the vectors 425GPD or p425TEF, resulting in the vectors p425-GPDHAP4 and p425-TEFHAP4 respectively. These shuttle vectors can self-replicate in E. coli and in yeast, but will only be maintained in yeast when selective pressure is present, i.e. when the recipient strain is LEU2 auxotrophic. In industrial strains without auxotrophic markers, integration of the GPDHAP or TEFHAP fusion is required to maintain the altered HAP gene stable in yeast. The promoter-HAP4 fragments were therefore recloned into the integration vector pKSPO2 (constructed at and provided by Gistbrocades). A 3359 bp Ecl136II-SalI GPDHAP4 fragment was isolated from p425-GPDHAP4 and a 3081 bp Ecl136II-SalI fragment was isolated from p425-TEFHAP4, which were cloned into pKSPO2 digested with SmaI and SalI. The different cloning steps are schematically drawn in FIG. 11. FIG. 12 shows the DNA sequence of the GPDHAP4 and TEFHAP4 fusions.

Transformation Procedures

Transformation of E. coli was carried out using the electroporation technique , using a Biorad E. coli pulser according to the description of the manufacturer.

Yeast cells were transformed according to the LiAc method described by Ito et al (29). Transformants of strain DL1 with the plasmid YCplac111::ADH1-HAP4 were selected on plates containing 2% glucose, 2% agar, 0.67% Yeast Nitrogen Base (Difco) supplied with the required aminoacids but lacking leucine. The industrial strains DS28911, DS18332 and DS19806 were plated on medium consisting of 1.8% nitrogen-free agar (Oxoid), 1.17% Yeast Carbon Base (Difco), 30 mM phosphte buffer pH 6.8 and 5 mM acetamide (Sigma).

Counterselection of Transformants

To select for transformants which after integration of the plasmid pKSPO2-GPDHAP4 or pKSPO2-TEFHAP4 recombine the plasmid sequences out of the genome (see FIG. 13), counterselection was carried out on plates containing 1.8% nitrogen-free agar, 1.17% Yeast Carbon Base, 30 mM phosphate buffer, 60 mM fluoro-acetamide (Fluka) and 0.1% (NH₄)₂SO₄. Transformants were grown in YPD medium for 60 to 70 generations by daily dilution of the cultures for 3 to 4 days. Aliqouts of the cultures were plated on the fluoro-acetamide containing plates, which selects for cells which have recombined out the acetamidase gene. The presence of the GPDHAP4 or TEFHAP4 fusion was tested by Southern blot analysis.

Growth of Yeast in Flask-batch Cultures

For shake flask cultivation, yeast cells were grown in either YPD (1% Yeast Extract, 1% BactoPeptone, 3% D-Glucose), YPEG (Yeast Extract , 1% BactoPeptone, 2% Ethanol, 2% Glycerol), YPL (lactate medium: 1.5% lactic acid, 2% Na-lactate, 0.1% Glucose, 8 mM MgSO4, 45 mM (NH)₂HPO₄, 0.5% Yeast-extract) or in mineral medium (0.67% Yeast Nitrogen Base) containing 3% D-glucose and supplemented with the appropiate aminoacids to obtain selective pressure for maintenaince of the transformed plasmid.

All cultures were inoculated from precultures which were prepared by inoculation of 5 ml medium with a colony from a plate. For northern analysis and oxygen consumption capacity measurements, the wild type and modified industrial yeast strains were precultured overnight in YPD and strain DL1 containing YCplac111::ADH1-HAP4 and Dl1 containing the empty YCplac111 vector were precultured overnight in mineral medium with additional amino acids. These cultures were used to inoculate 100 ml YPEG and/or YPD medium and grown overnight at 28° C. to OD₆₀₀˜1.5, after which cells were harvested by centrifugation.

Growth of Yeast in Fermentor-batch Cultures

Transformed yeast cells were grown in selective mineral Evans medium containing 30 g l⁻¹ D-Glucose and supplemented with 40 mg l⁻¹ uracil and L-histidine. The mineral medium contained: NaH₂PO₄.2H₂O, 10 mM; KCl, 10 mM; MgCl₂.6H₂O, 1.25 mM; NH₄Cl, 0.1 mM; Na₂SO₄, 2 mM; C₆H₉NO₆, 2 mM; CaCl₂, 20 mM; ZnO, 25.3 mM; FeCl₃.H₂O, 99.9 mM; MnCl₂, 50.5 mM; CuCl₂, 5 mM; CoCl₂, 10 mM; H₃BO₃, 5.2 mM; Na₂MoO₄.2H₂O, 0.08 mM. After heat sterilization of the medium at 120° C., filter sterilized vitamins were added to final concentrations per liter of myoinositol, 0.55 mM; nicotineacid, 0.16 mM; Ca-D(+)panthothenate, 0.02 mM; pyridoxine-HCL, 0.013 mM; thiamine-HCl, 0.006 mM; biotin, 0.02 mM. Cultivation was performed at 28° C. in New Brunswick Scientific Bioflow fermentors, at a stirrer speed of 900 rpm. The pH was kept constant at pHS.0 via the automatic addition of 2 mol l⁻¹ NaOH. Antifoam (BDH) was added automatically at fixed time intervals. The fermentors were flushed with air at a flow rate of 0.8 I min⁻¹. The starting working volume of the cultures was 1.0 or 1.4 liter. Samples of 30 ml were taken every hour for analysis of culture purity, dry weight, substrate consumption and product formation.

Determination of Culture Optical Density and Dry Weight

Optical cell density of cultures was measured in a spectrophotometer at 600nm. The dry weight of cultures was determined by centrifugation of 10.0 ml of culture, washing cells with demineralized H₂O, and drying the cellpellet overnight at 80° C. Parallel samples varied by less than 1%.

Substrate Consumption and Product Formation in Liquid Medium

Concentrations of carbon compounds, like glucose, ethanol, glycerol, acetate and pyruvate were determined by HPLC analysis using an Aminex HPX87H organic acids column of Biorad at 65° C. The column was eluted with 5 mM H₂SO₄. Detection was by means of a 2142 refractive index detector (LKB) and SP4270 integrator of SpectraPhysics.

Analysis of CO₂ Production and O₂ Consumption

During cultivation in fermentors, the dissolved carbondioxide concentration was continuously monitored by a cervomex IR PA404 gas analyzer and oxygen by a Taylor cervomex OA 272 gas analyzer. The absolute amounts of gas consumption/production during the time course of the experiment was calculated by the mean of the gas concentration, corrected for the decreasing volume of the culture.

For oxygen consumption capacity measurements of flask-batch grown cells, the cells were harvested, washed three times with ice-cold demineralized H₂O, and resuspended in oxygraph buffer (1% Yeast Extract, 0.1% KH₂PO₄, 0.12% (NH4)₂SO₄, pH 4.5) at 200 OD units ml⁻¹. Oxygen consumption capacity of the cells was measured with a Clark electrode, with 0.1 mM ethanol as substrate.

RNA Isolation, Northern Analysis and Labelling of DNA Fragments

Cells were harvested and RNA was isolated, separated on a non-denaturing 1.2% agarose gel and transferred to a nitrocellulose filter as described by De Winde (30). Prehybridization was performed in hybridization buffer containing 50 microgram/ml single stranded salm sperm DNA (50% formamide, 25 mM NaPi pH 6.5, 5×SSc, 5×Denhardt, as described by Maniatis (31). DNA fragments used as probes in this study include a 840 bp HindIII-SalI fragment from pJH1 (30); a 1.6 kb BamHI-KpnI fragment containing the yeast actin gene (24); a 2.5 kb HindIII-SalI fragment from YE23SH containing the QCR2 gene (25); a 1333 bp NcoI-HindIII fragment from pAZ6 containing the yeast PDA1 gene (24) and a 1.2 kb BamHI-HindIII fragment from YE23R-SOD/SUC containing the SUC2 gene (26). Fragments were labeled ³²p by nicktranslation as described by Maniatis et al. Labeled probes were added to the prehybridization buffer and hybridization was performed overnight at 42° C. Blots were washed once with 2×SSC 0.1% SDS, twice with 1×SSC, 0.1% SDS and finally with 0.5×SSC, 0.1% SDS. Blots were air-dried completely and autoradiography was performed with Kodak Xomat 100 film or analysed by a Storm 840 Molecular Dynamics Phosphorimager.

Chromosomal DNA Isolation and Southern Blotting

Chromosomal DNA was isolated according to the method described by Hoffman and Winston (34). 10 microliter of the chromosomal DNA was digested with either BamHI or EcoRI. The digested DNA was separated on a 1% agarose gel and transferred to nitrocellulose filter as described in Maniatis (31) Prehybridization of the filters was performed at 65° C. in 6×SSC, 5×Denhardt, 0.5% SDS and 100 microgram/ml salmon sperm DNA. After 4 hours prehybridization, a radioactive labeled KpnI-XbaI fragment from pKSPO2-GPDHAP (see FIGS. 11B and 13) was added and hybridization was continued overnight at 65° C. Blots were washed once with 2×SSC 0.1% SDS, twice with 1×SSC, 0.1% SDS and finally with 0.5×SSC, 0.1% SDS. Blots were air-dried completely and radioactivity was visualised and analysed by a Storm 840 Molecular Dynamics Phosphorimager.

DESCRIPTION OF THE FIGURES

FIG. 1. Simplified view of the carbon metabolism in glucose-repressed Saccharomyces cerevisiae cells. Only a number of intermediates are shown, and specific pathways for the utilization of other carbon sources than glucose are only shown for maltose and galactose.

FIG. 2. Transcriptional regulation of a typical yeast promoter. ATG denotes the start codon of the corresponding translational open reading frame. Abbreviations: UIS, upstream induction site; UAS, upstream activation site; URS, upstream repressor site; T, TATA-box, I, initiation site.

FIG. 3. Schematic representation of the regulatory pathways involved in glucose repression of a number of genes in yeast. Activating functions are denoted as (+), repressing/inactivating functions as (−), and interactions which are not resolved yet are denoted as (?).

FIG. 4. Nucleotide and amino-acid sequence of the HAP2 gene (SEQ ID NO:1).

FIG. 5. Nucleotide and amino-acid sequence of the HAP3 gene (SEQ ID NO:3).

FIG. 6. Nucleotide and amino-acid sequence of the HAP4 gene (SEQ ID NO:5).

FIG. 7. Illustrates the construction of YCplac111::ADH1-HAP4, a yeast shuttle vector where HAP4 is expressed from the ADH1 promoter. Plasmids are not drawn to scale.

FIG. 8. Nucleotide and amino-acid sequence of the ADH1-HAP4 fusion (SEQ ID NO:7).

FIG. 9. Northern blot analysis of HAP4 overexpression. Dl1 containing YCplac111ADH1 (WT) and DL1 containing YCplac111::ADH1-HAP4 (+HAP4) were grown in medium containing 2% glucose (D) or 2% ethanol/2% glycerol (EG). Total RNA was hybridized with probes specific for HAP4, actin (ACT), QCR8, or SUC2 mRNA.

FIG. 10. Biomass yield. Dl1 containing YCplac111::ADH1 (DL1) and DL1 containing YCplac111::ADH1-HAP4 (DL1HAP) were grown in fermentors. Samples for determination of dry weight and glucose concentration were taken with an interval of one hour.

FIGS. 11. 11A. Construction of the plasmids p425-GPDHAP4, p425-TEFHAP4, 11B. Construction of the plasmids pKSPO2-GPDHAP4 and pKSPO2-TEFHAP4

FIGS. 12. 12A. Nucleotide and amino acid sequence of the GPD1 promoter fused to the coding region of HAP4 (SEQ ID NO:8). 12B. Nucleotide and amino acid sequence of the TEF2 promoter fused to the coding region of HAP4 (SEQ ID NO:9). The fragments shown are the fragments as cloned into pKSPO2 and represent the sequence as integrated in the genome

FIG. 13. Scheme of genomic DNA at HAP4 and SIT locus and chromosomal rearrangements after integration of the plasmid pKSPO2-GPD1 or pKSPO2-TEFHAP4 on either the SIT2 locus or the HAP4 locus, and after counterselection on fluoro-acetamide (Fac). Fragments generated after digestion with BamHI or EcoRI which hybridize with the KpnI-XbaI probe (shown as thick black bar) are visualized for comparison with the Southern blots shown in FIGS. 13 and 15. SITpr: SIT2 promoter, HAP4pr: HAP4 promoter, G/Tpr: GPD or TEF promoter, B: BamHI, E: EcoRI, K: KpnI, X: XbaI, FacR: fluoro-acetamide resistant.

FIG. 14. Southern blot of chromosomal DNA digested with BamHI of transformants with pKSPO2-GPDHAP (GH) or pKSPO2-TEFHAP (TH) integrated at the HAP4 locus. The blot was hybridized with the KpnI-XbaI probe shown in FIG. 12, visualizing fragments containing SIT2 and/or HAP4 sequences. Radioactivity was visualised and analysed by a Storm 840 Molecular Dynamics Phosphorimager.

FIG. 15. Northern blot of total mRNA of transformants with pKSPO2-GPDHAP (GH) or pKSPO2-TEFHAP (TH) integrated at the HAP4 locus as in FIG. 14. The blots were hybridized with probes specific for HAP4, QCR8 or PDA1.

FIGS. 16A and 16B. Southern blot of chromosomal DNA digested with BamHI or EcoRI of transformants containing pKSPO2-GPDHAP (GH) or pKSPO2-TEFHAP (TH) integrated at the SIT2 locus 16(A) and three clean transformants containing the GPDHAP4 or TEFHAP4 fusion after counterselection on fluoro-acetamide 16(B). The blot was hybridized with the KpnI-XbaI probe shown in FIG. 12, visualizing fragments containing SIT2 and/or HAP4 sequences.

FIGS. 17A. and 17B. Northern blot of total mRNA of transformants with pKSPO2-GPDHAP (GE) or pKSPO2-TEFHAP (TH) integrated at the SIT2 locus as in FIG. 16. The blots were hybridized with probes specific for HAP4, QCR8 or PDA1 mRNA.

REFERENCES

1. Ronne, H: Glucose repression in fungi. (1995) Trends in Genet. 11: p. 12

2. Thevelein, J. M., Hohmann, S.(1995) Trends in Biochem. Sci. 20: p. 3

3. Sierkstra, L. N., Nouwen, N. P., Verbakel, J. M. A., Verrips, C. T. (1993) Yeast 9: p. 787

4. Needleman, R. B., Kaback, D. B., Dubin, R. A., Perkins, E. L., Rosenberg, N. G., Sutherland, K. A., Forrest, D. B., Michels, C. A. (1984) Proc. Natl. Acad. Sci. USA 81, p2811

5. Lesage, P., Yand, X., Carlson, M.(1996) Molec. Cell. Biol. 16: p. 1921

6. Nehlin, J. O. and Ronne, H. (1990) EMBO J. 9: p. 2891

7. Treitel, M. A., Carlson, M. (1995) Proc. Natl. Acad. Sci., USA 92: p. 3132

8. Guarente, L., Lalonde, B., Gifford, P., Alani, E.(1984) Cell , 36: p. 503

9. de Winde, J. H., Grivell, L. A., ed. Global regulation of mitochondrial biogenesis in Saccaromyces cerevisiae. Progr. in Nucl. Acid Res. Mol. Biol., ed. Cohn, W. E., Moldave, K. Vol. 46. 1993, Academic Press: San Diego. p. 51

10. Dang, V. D., Valens, M., Bolotin-Fukuhara, M., Daignan-Fornier, B. (1994). Yeast 10: p. 1273

11. Bowman, S. B., Zaman, Z., Collinson, L. P., Brown, A. J. P., Dawes, I. W. (1992) Mol. Gen. Genet. 231: p. 296

12. Rozenkrantz, M., Kell, C. S., Pennel, E. A., Devenish, L. J. (1994) Mol. Microbiol. 13: p. 11913.

13. Forsburg, S., Guarente, L. (1989) Genes and Dev. 3: p. 1166

14. Olesen, J., Hahn, S., Guarente, L.(1987) Cell SI: p. 953

15. Li, X. Y., Mantovani, R., Hooft van Huijsduijnen, R., Andre, I., Benoist, C., Mathis, D.(1992) Nucl. Acids Res. 20: p. 1087

16. Sinha, S., Maity, S. N., Lu, J., de Crombrugghe, B.(1995) Proc. Natl. Acad. Sci., USA, 92: p. 1624

17. Johnston, M. and Carlson, M. (1992) In Jones, J., Pringle, J. R., Broach, J. R. (ed.) The molecular and cellular biology of the yeast Saccharomyces: gene expression. Cold. Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p. 220-237

18. Trumbly, R. J. (1992) Molec. Microbiol. 6: p. 15

19. Klein, C. J. L., Olsson, L., Ronnow, B., Mikkelsen, J. D., Nielsen, J. (1996) Applied and Environmental Microbiology 62: p 4441

20. Gottlin-Ninga, E., Kaback, D. B. (1986) Mol. Cell. Biol. 6: p. 2185

21. Rothstein, R. J. (1983) Methods in Enzymology 101, p202

22. Mümberg, D., Muller, R., Funk, M. (1995) Gene 156: p. 119

23. Brown, T. A., Trumpower, B. L. (1995) J. Bact. 177: p. 1380

24. Wenzel, T. J., Zuurmond, A.-M., Bergmans, A., van den Berg, J. A., Steensma, H. Y. (1994) Yeast 10: p. 297

25. Gietz, R. D. & Sugino,A. (1988) Gene 74: p. 527

26. van Loon, A. P. G. M., Brändli, A. W., Pesold-Hurt, B., Blank, D., Schatz, G. (1987) EMBO J. 6: p. 2433

27. Hurt, E. C., Müller, U., Schatz, G. (1985) EMBO J. 4: p. 3509

28. Mc Knight, G. L. and McConaughy, B. L. (1983) Proc. Natl. Acad. Sci. USA 80: p. 4412

29. H. Ito, Y. Fukuda, M. Murata, A. Kimura (1983), J. Bact. 153: p. 163

30. De Winde, J. H. and Grivell, L. A.(1992) Mol.Cell. Biol 12: p. 2872.

31. Maniatis et. al.(1982) Molecular Cloning, A Laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p 9.49-9.52)

32. Ng, R. and Abelson, J. (1980) Proc. Natl. Acad. Sci. USA 77: p. 3912

33. Oudshoorn, P., van Steeg, H., Swinkels, B., Schoppink, P., Grivell, L. A. (1987) Eur.J. Biochem. 163: p. 97

34. van Steeg, H., Oudshoorn, P., van Hell, B., Polman, J. E. M., Grivell, L. A. (1986) EMBO J. 5: p. 3643

35. Hoffman, C. S., Winston, F. (1987) Gene 57: p. 267

TABLE I Effect of induced expression of HAP4 on transcription expression functional expression in HAP 4 in HAP 4 - binding wildtype overproducer gene protein site on glucose on glucose QCR8 11 kD subunit + repressed induced VIII QCR7 14 kD subunit + repressed induced VII QCR2 40 kD subunit + repressed induced II CYC1 iso-1- + repressed induced cytochrome c SUC2 invertase − repressed repressed

TABLE II Oxygen consumption capacity of D11 and DL1HAP yeast cells oxygen consumption nmol/min/mg dry weight Glucose (4%)-grown cells Lactate-grown cells DL1 9.4 88.1 (9.4 × wt Glu) DL1HAP 18.1 (1.9 × wt Glu) 86.7 (9.2 × wt Glu)

TABLE III Biomass yield. ^(Y)glu strain g. dry weight · g⁻¹Glu Dl1 10.1 DL1HAP 14.8

TABLE IV Carbon compounds in culture medium during batch growth in fermentors. Data are mean values of several experiments ethanol acetate glycerol strain mol.L⁻¹.g⁻¹ dry weight cells Dl1 79.5 1.6 10.2 DL1HAP 49.4 3.6 2.9

TABLE V Carbon fluxes indicated as percentage mol C of consumed glucose Carbon balance DL1 = 103%, DL1HAP = 108% strain CO_(2-TCA) ethanol acetate glycerol biomass Dl1 7.3 72.5 1.4 9.5 12.2 DL1HAP 15.5 66.0 5.0 4.0 17.8

TABLE VI Oxygen consumption capacity of industrial strains oxygen consumption nmol/min/mg dry weight Glucose (4%)-grown cells Lactate-grown cells DS28911 25.3 ND DS28911-GH2a 104.0 (4.1 × wt glu) ND DS28911-TH2a 75.0 (3.0 × wt glu) ND DS18332 31.5 241 (7.7 × wt glu) DS18332-GH1a 94.5 (3.0 × wt glu) ND DS18332-TH2a 70.3 (2.2 × wt glu) ND DS19806 23.8 ND DS19806-GH1a 87.2 (3.7 × wt glu) ND DS19806-TH2a 65.6 (2.8 × wt glu) ND

9 1 1198 DNA Artificial Sequence Description of Artificial Sequence DNA construct encoding HAP2 1 tcgttattta gcaatcttac ccggaaaact tcttgtatcg taacattaat tctcccttac 60 aaggggacct cgatgaataa ataaagtgtt tgtgttgaag gtacttgcaa aagggcaact 120 cactgtcgtt tataatttga tttactaatc gcatctgtat ttggaaaagc atttcttttt 180 ggaagaggaa caagaacgcc atg tca gca gac gaa acg gat gcg aaa ttt cat 233 Met Ser Ala Asp Glu Thr Asp Ala Lys Phe His 1 5 10 cca tta gaa aca gat ctg caa tct gat aca gcg gct gca aca tca acg 281 Pro Leu Glu Thr Asp Leu Gln Ser Asp Thr Ala Ala Ala Thr Ser Thr 15 20 25 gca gca gct tca cgc agt ccc tct ctt caa gag aag ccc ata gag atg 329 Ala Ala Ala Ser Arg Ser Pro Ser Leu Gln Glu Lys Pro Ile Glu Met 30 35 40 ccc ttg gat atg gga aaa gcg cct tct cca aga ggc gaa gat caa cgg 377 Pro Leu Asp Met Gly Lys Ala Pro Ser Pro Arg Gly Glu Asp Gln Arg 45 50 55 gtt aca aat gaa gaa gat ttg ttt ttg ttt aac aga ttg cgg gca tca 425 Val Thr Asn Glu Glu Asp Leu Phe Leu Phe Asn Arg Leu Arg Ala Ser 60 65 70 75 cag aat aga gtt atg gac tcc ttg gaa cca caa caa cag tca cag tat 473 Gln Asn Arg Val Met Asp Ser Leu Glu Pro Gln Gln Gln Ser Gln Tyr 80 85 90 aca tct tcc agt gtc agt acg atg gaa cca tct gcc gac ttt act agt 521 Thr Ser Ser Ser Val Ser Thr Met Glu Pro Ser Ala Asp Phe Thr Ser 95 100 105 ttc tct gca gtg act act tta ccg cct cct cct cat caa caa caa cag 569 Phe Ser Ala Val Thr Thr Leu Pro Pro Pro Pro His Gln Gln Gln Gln 110 115 120 caa caa cag cag cag cag cag cag cag caa ttg gtg gtt caa gcc cag 617 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Val Val Gln Ala Gln 125 130 135 tac acc caa aat caa cca aac ttg caa agc gat gtt tta gga acc gct 665 Tyr Thr Gln Asn Gln Pro Asn Leu Gln Ser Asp Val Leu Gly Thr Ala 140 145 150 155 ata gca gag caa cca ttt tat gtt aat gcc aag cag tac tac cga att 713 Ile Ala Glu Gln Pro Phe Tyr Val Asn Ala Lys Gln Tyr Tyr Arg Ile 160 165 170 ttg aaa agg cga tat gca aga gct aaa cta gag gaa aag cta cga ata 761 Leu Lys Arg Arg Tyr Ala Arg Ala Lys Leu Glu Glu Lys Leu Arg Ile 175 180 185 tca aga gaa cga aag cca tac tta cac gaa tct cga cat aaa cat gcg 809 Ser Arg Glu Arg Lys Pro Tyr Leu His Glu Ser Arg His Lys His Ala 190 195 200 atg cga aga cct cgt ggt gaa ggt ggg agg ttc ttg aca gcc gct gag 857 Met Arg Arg Pro Arg Gly Glu Gly Gly Arg Phe Leu Thr Ala Ala Glu 205 210 215 atc aaa gcc atg aaa tcg aag aaa agt ggg gct agc gat gat cct gac 905 Ile Lys Ala Met Lys Ser Lys Lys Ser Gly Ala Ser Asp Asp Pro Asp 220 225 230 235 gat agt cat gag gat aaa aaa atc act act aaa ata ata caa gaa cag 953 Asp Ser His Glu Asp Lys Lys Ile Thr Thr Lys Ile Ile Gln Glu Gln 240 245 250 ccg cat gct act tcc acc gca gct gca gca gac aaa aaa aca 995 Pro His Ala Thr Ser Thr Ala Ala Ala Ala Asp Lys Lys Thr 255 260 265 taattttgta atattccaat gttaatatca ttcctaaaag aactaaaagt gccctcttat 1055 accacatggt atccatatgg cctatttaat ctgaatcaat atgtatatgt acttttacca 1115 atctcgtttc gtttcgtttc gtttcatttc taacagacct atgtactccg ctggaaaaga 1175 aaccatattg cgatcgtatt tac 1198 2 265 PRT Artificial Sequence Description of Artificial Sequence HAP2 2 Met Ser Ala Asp Glu Thr Asp Ala Lys Phe His Pro Leu Glu Thr Asp 1 5 10 15 Leu Gln Ser Asp Thr Ala Ala Ala Thr Ser Thr Ala Ala Ala Ser Arg 20 25 30 Ser Pro Ser Leu Gln Glu Lys Pro Ile Glu Met Pro Leu Asp Met Gly 35 40 45 Lys Ala Pro Ser Pro Arg Gly Glu Asp Gln Arg Val Thr Asn Glu Glu 50 55 60 Asp Leu Phe Leu Phe Asn Arg Leu Arg Ala Ser Gln Asn Arg Val Met 65 70 75 80 Asp Ser Leu Glu Pro Gln Gln Gln Ser Gln Tyr Thr Ser Ser Ser Val 85 90 95 Ser Thr Met Glu Pro Ser Ala Asp Phe Thr Ser Phe Ser Ala Val Thr 100 105 110 Thr Leu Pro Pro Pro Pro His Gln Gln Gln Gln Gln Gln Gln Gln Gln 115 120 125 Gln Gln Gln Gln Gln Leu Val Val Gln Ala Gln Tyr Thr Gln Asn Gln 130 135 140 Pro Asn Leu Gln Ser Asp Val Leu Gly Thr Ala Ile Ala Glu Gln Pro 145 150 155 160 Phe Tyr Val Asn Ala Lys Gln Tyr Tyr Arg Ile Leu Lys Arg Arg Tyr 165 170 175 Ala Arg Ala Lys Leu Glu Glu Lys Leu Arg Ile Ser Arg Glu Arg Lys 180 185 190 Pro Tyr Leu His Glu Ser Arg His Lys His Ala Met Arg Arg Pro Arg 195 200 205 Gly Glu Gly Gly Arg Phe Leu Thr Ala Ala Glu Ile Lys Ala Met Lys 210 215 220 Ser Lys Lys Ser Gly Ala Ser Asp Asp Pro Asp Asp Ser His Glu Asp 225 230 235 240 Lys Lys Ile Thr Thr Lys Ile Ile Gln Glu Gln Pro His Ala Thr Ser 245 250 255 Thr Ala Ala Ala Ala Asp Lys Lys Thr 260 265 3 835 DNA Artificial Sequence Description of Artificial Sequence DNA construct encoding HAP3 3 caaaccttct gccaaaatat agcacaatag aagtaccata ttacgttcga tgccacgaca 60 atatcgcgct acgtgcgttt tttggtccgc tctttcagac taagtaaaaa aagagctgcg 120 aatagtagct ttccgccaat caaactcaag agcaggacta agctagatag taacacaagt 180 ggcacaaacc tctcgagaat atg aat acc aac gag tcc gaa cat gtt agc aca 233 Met Asn Thr Asn Glu Ser Glu His Val Ser Thr 1 5 10 agc cca gag gat act cag gag aac ggt gga aac gct agc tcc agc ggc 281 Ser Pro Glu Asp Thr Gln Glu Asn Gly Gly Asn Ala Ser Ser Ser Gly 15 20 25 agt ttg cag caa att tcc acg cta aga gag cag gac aga tgg cta ccc 329 Ser Leu Gln Gln Ile Ser Thr Leu Arg Glu Gln Asp Arg Trp Leu Pro 30 35 40 atc aac aat gta gcg cga ctc atg aag aat act ctc cca ccg agt gct 377 Ile Asn Asn Val Ala Arg Leu Met Lys Asn Thr Leu Pro Pro Ser Ala 45 50 55 aag gta tcg aaa gat gcg aaa gag tgc atg cag gag tgt gtc agt gag 425 Lys Val Ser Lys Asp Ala Lys Glu Cys Met Gln Glu Cys Val Ser Glu 60 65 70 75 ctc att tct ttt gtg act agc gag gcc agc gat cga tgc gct gct gac 473 Leu Ile Ser Phe Val Thr Ser Glu Ala Ser Asp Arg Cys Ala Ala Asp 80 85 90 aaa aga aag acg ata aac ggg gaa gac att ctc ata tca ttg cac gcc 521 Lys Arg Lys Thr Ile Asn Gly Glu Asp Ile Leu Ile Ser Leu His Ala 95 100 105 tta gga ttc gag aac tat gca gag gtg ttg aaa atc tac ttg gct aaa 569 Leu Gly Phe Glu Asn Tyr Ala Glu Val Leu Lys Ile Tyr Leu Ala Lys 110 115 120 tac agg caa caa cag gcg ctg aag aat caa cta atg tat gag cag gac 617 Tyr Arg Gln Gln Gln Ala Leu Lys Asn Gln Leu Met Tyr Glu Gln Asp 125 130 135 gac gaa gag gtg cct tgagaagaca aaaccaggtg gtagatcgca aaagttgcta 672 Asp Glu Glu Val Pro 140 gctgtcagga tggaatagca cggggctatt tcctgctggt cgttggttct cgtgtaatta 732 atgaatgtaa cgatatagat aatattttat tgttagtgtg taatgtattc aatgtaatgt 792 atgggtgctt tgtaaagggt gtatgatgtt tgccaccgga agg 835 4 144 PRT Artificial Sequence Description of Artificial Sequence HAP3 4 Met Asn Thr Asn Glu Ser Glu His Val Ser Thr Ser Pro Glu Asp Thr 1 5 10 15 Gln Glu Asn Gly Gly Asn Ala Ser Ser Ser Gly Ser Leu Gln Gln Ile 20 25 30 Ser Thr Leu Arg Glu Gln Asp Arg Trp Leu Pro Ile Asn Asn Val Ala 35 40 45 Arg Leu Met Lys Asn Thr Leu Pro Pro Ser Ala Lys Val Ser Lys Asp 50 55 60 Ala Lys Glu Cys Met Gln Glu Cys Val Ser Glu Leu Ile Ser Phe Val 65 70 75 80 Thr Ser Glu Ala Ser Asp Arg Cys Ala Ala Asp Lys Arg Lys Thr Ile 85 90 95 Asn Gly Glu Asp Ile Leu Ile Ser Leu His Ala Leu Gly Phe Glu Asn 100 105 110 Tyr Ala Glu Val Leu Lys Ile Tyr Leu Ala Lys Tyr Arg Gln Gln Gln 115 120 125 Ala Leu Lys Asn Gln Leu Met Tyr Glu Gln Asp Asp Glu Glu Val Pro 130 135 140 5 2065 DNA Artificial Sequence Description of Artificial Sequence DNA construct encoding HAP4 5 taaaggaacc agaaaaataa aaaagggtca ttatttattt gagcagatca ttatcaaacg 60 cataggaaga gaaaaaacac agttttattt tttttccaca catatttatt ggtctcctag 120 tacatcaaag agcattttaa tgggttgctg atttgtttta cctacatttt ctagtacaaa 180 aaaaaaacaa aaaaagaatc atg acc gca aag act ttt cta cta cag gcc tcc 233 Met Thr Ala Lys Thr Phe Leu Leu Gln Ala Ser 1 5 10 6 554 PRT Artificial Sequence Description of Artificial Sequence HAP4 6 Met Thr Ala Lys Thr Phe Leu Leu Gln Ala Ser Ala Ser Arg Pro Arg 1 5 10 15 Ser Asn His Phe Lys Asn Glu His Asn Asn Ile Pro Leu Ala Pro Val 20 25 30 Pro Ile Ala Pro Asn Thr Asn His His Asn Asn Ser Ser Leu Glu Phe 35 40 45 Glu Asn Asp Gly Ser Lys Lys Lys Lys Lys Ser Ser Leu Val Val Arg 50 55 60 Thr Ser Lys His Trp Val Leu Pro Pro Arg Pro Arg Pro Gly Arg Arg 65 70 75 80 Ser Ser Ser His Asn Thr Leu Pro Ala Asn Asn Thr Asn Asn Ile Leu 85 90 95 Asn Val Gly Pro Asn Ser Arg Asn Ser Ser Asn Asn Asn Asn Asn Asn 100 105 110 Asn Ile Ile Ser Asn Arg Lys Gln Ala Ser Lys Glu Lys Arg Lys Ile 115 120 125 Pro Arg His Ile Gln Thr Ile Asp Glu Lys Leu Ile Asn Asp Ser Asn 130 135 140 Tyr Leu Ala Phe Leu Lys Phe Asp Asp Leu Glu Asn Glu Lys Phe His 145 150 155 160 Ser Ser Ala Ser Ser Ile Ser Ser Pro Ser Tyr Ser Ser Pro Ser Phe 165 170 175 Ser Ser Tyr Arg Asn Arg Lys Lys Ser Glu Phe Met Asp Asp Glu Ser 180 185 190 Cys Thr Asp Val Glu Thr Ile Ala Ala His Asn Ser Leu Leu Thr Lys 195 200 205 Asn His His Ile Asp Ser Ser Ser Asn Val His Ala Pro Pro Thr Lys 210 215 220 Lys Ser Lys Leu Asn Asp Phe Asp Leu Leu Ser Leu Ser Ser Thr Ser 225 230 235 240 Ser Ser Ala Thr Pro Val Pro Gln Leu Thr Lys Asp Leu Asn Met Asn 245 250 255 Leu Asn Phe His Lys Ile Pro His Lys Ala Ser Phe Pro Asp Ser Pro 260 265 270 Ala Asp Phe Ser Pro Ala Asp Ser Val Ser Leu Ile Arg Asn His Ser 275 280 285 Leu Pro Thr Asn Leu Gln Val Lys Asp Lys Ile Glu Asp Leu Asn Glu 290 295 300 Ile Lys Phe Phe Asn Asp Phe Glu Lys Leu Glu Phe Phe Asn Lys Tyr 305 310 315 320 Ala Lys Val Asn Thr Asn Asn Asp Val Asn Glu Asn Asn Asp Leu Trp 325 330 335 Asn Ser Tyr Leu Gln Ser Met Asp Asp Thr Thr Gly Lys Asn Ser Gly 340 345 350 Asn Tyr Gln Gln Val Asp Asn Asp Asp Asn Met Ser Leu Leu Asn Leu 355 360 365 Pro Ile Leu Glu Glu Thr Val Ser Ser Gly Gln Asp Asp Lys Val Glu 370 375 380 Pro Asp Glu Glu Asp Ile Trp Asn Tyr Leu Pro Ser Ser Ser Ser Gln 385 390 395 400 Gln Glu Asp Ser Ser Arg Ala Leu Lys Lys Asn Thr Asn Ser Glu Lys 405 410 415 Ala Asn Ile Gln Ala Lys Asn Asp Glu Thr Tyr Leu Phe Leu Gln Asp 420 425 430 Gln Asp Glu Ser Ala Asp Ser His His His Asp Glu Leu Gly Ser Glu 435 440 445 Ile Thr Leu Ala Asp Asn Lys Phe Ser Tyr Leu Pro Pro Thr Leu Glu 450 455 460 Glu Leu Met Glu Glu Gln Asp Cys Asn Asn Gly Arg Ser Phe Lys Asn 465 470 475 480 Phe Met Phe Ser Asn Asp Thr Gly Ile Asp Gly Ser Ala Gly Thr Asp 485 490 495 Asp Asp Tyr Thr Lys Val Leu Lys Ser Lys Lys Ile Ser Thr Ser Lys 500 505 510 Ser Asn Ala Asn Leu Tyr Asp Leu Asn Asp Asn Asn Asn Asp Ala Thr 515 520 525 Ala Thr Asn Glu Leu Asp Gln Ser Ser Phe Ile Asp Asp Leu Asp Glu 530 535 540 Asp Val Asp Phe Leu Lys Val Gln Val Phe 545 550 7 2394 DNA Artificial Sequence Description of Artificial Sequence DNA construct consisting of the ADH1 promoter fused to the coding region of HAP4 7 atccttttgt tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca 60 tacatcggga ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga 120 gatatacaat agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt 180 acactgcctc attgatggtg gtacataacg aactaatact gtagccctag acttgatagc 240 catcatcata tcgaagtttc actacccttt ttccatttgc catctattga agtaataata 300 ggcgcatgca acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca 360 tatccgcaat gacaaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac 420 agcaccaaca gatgtcgttg ttccagagct gatgaggggt atcttcgaac acacgaaact 480 ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt 540 ccagttactt gaatttgaaa taaaaaaagt ttgccgcttt gctatcaagt ataaatagac 600 ctgcaattat taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc 660 tttttctgca caatatttca agctatacca agcatacaat caaggaattc gagctcgccc 720 catgaccgca aagacttttc tactacaggc ctccgctagt cgccctcgta gtaaccattt 780 taaaaatgag cataataata ttccattggc gcctgtaccg atcgccccaa ataccaacca 840 tcataacaat agttcgctgg aattcgaaaa cgatggcagt aaaaagaaga agaagtctag 900 cttggtggtt agaacttcaa aacattgggt tttgccccca agaccaagac ctggtagaag 960 atcatcttct cacaacactc tacctgccaa caacaccaat aatattttaa atgttggccc 1020 taacagcagg aacagtagta ataataataa taataataac atcatttcga ataggaaaca 1080 agcttccaaa gaaaagagga aaataccaag acatatccag acaatcgatg aaaagctaat 1140 aaacgactcg aattacctcg catttttgaa gttcgatgac ttggaaaatg aaaagtttcg 1200 ttcttctgcc tcctccattt catctccatc ttattcatct ccatcttttt caagttatag 1260 aaatagaaaa aaatcagaat tcatggacga tgaaagctgc accgatgtgg aaaccattgc 1320 tgctcacaac agtctgctaa caaaaaacca tcatatagat tcttcttcaa atgttcacgc 1380 accacccacg aaaaaatcaa agttgaacga ctttgattta ttgtccttat cttccacatc 1440 ttcatcggcc actccggtcc cacagttgac aaaagatttg aacatgaacc taaattttca 1500 taagatccct cataaggctt cattccctga ttctccagca gatttctctc cagcagattc 1560 agtctcgttg attagaaacc actccttgcc tactaatttg caagttaagg acaaaattga 1620 ggatttgaac gagattaaat tctttaacga tttcgagaaa cttgagtttt tcaataagta 1680 tgccaaagtc aacacgaata acgacgttaa cgaaaataat gatctctgga attcttactt 1740 acagtctatg gacgatacaa caggtaagaa cagtggcaat taccaacaag tggacaatga 1800 cgataatatg tctttattga atctgccaat tttggaggaa accgtatctt cagggcaaga 1860 tgataaggtt gagccagatg aagaagacat ttggaattat ttaccaagtt caagttcaca 1920 acaagaagat tcatcacgtg ctttgaaaaa aaatactaat tctgagaagg cgaacatcca 1980 agcaaagaac gatgaaacct atctgtttct tcaggatcag gatgaaagcg ctgattcgca 2040 tcaccatgac gagttaggtt cagaaatcac tttggctgac aataagtttt cttatttgcc 2100 cccaactcta gaagagttga tggaagagca ggactgtaac aatggcagat cttttaaaaa 2160 tttcatgttt tccaacgata ccggtattga cggtagtgcc ggtactgatg acgactacac 2220 caaagttctg aaatccaaaa aaatttctac gtcgaagtcg aacgctaacc tttatgactt 2280 aaacgataac aacaatgatg caactgccac caatgaactt gatcaaagca gtttcatcga 2340 cgaccttgac gaagatgtcg attttttaaa ggtacaagta ttttaagggg atcc 2394 8 3360 DNA Artificial Sequence Description of Artificial Sequence DNA construct consisting of the GPD1 promoter fused to the coding region of HAP4 8 gagctctggg gtttgagcaa gtctaagttt acgtagcata aaaattctcg gattgcgtca 60 aataataaaa aaagtaaccc cacttctact tctacatcgg aaaaacattc cattcacata 120 tcgtctttgg cctatcttgt tttgtcctcg gtagatcagg tcagtacaaa cgcaacacga 180 aagaacaaaa aaagaagaaa acagaaggcc aagacagggt caatgagact gttgtcctcc 240 tactgtccct atgtctctgg ccgatcacgc gccattgtcc ctcagaaaca aatcaaacac 300 ccacaccccg ggcacccaaa gtccccaccc acaccaccaa tacgtaaacg gggcgccccc 360 tgcaggccct cctgcgcgcg gcctcccgcc ttgcttctct ccccttcctt ttctttttcc 420 agttttccct attttgtccc tttttccgca caacaagtat cagaatgggt tcatcaaatc 480 tatccaacct aattcgcacg tagactggct tggtattggc agtttcgtag ttatatatat 540 actaccatga gtgaaactgt tacgttacct taaattcttt ctccctttaa ttttctttta 600 tcttactctc ctacataaga catcaagaaa caattgtata ttgtacaccc cccccctcca 660 caaacacaaa tattgataat ataaagtcta gaactagtgg atccccccat gaccgcaaag 720 acttttctac tacaggcctc cgctagtcgc cctcgtagta accattttaa aaatgagcat 780 aataatattc cattggcgcc tgtaccgatc gccccaaata ccaaccatca taacaatagt 840 tcgctggaat tcgaaaacga tggcagtaaa aagaagaaga agtctagctt ggtggttaga 900 acttcaaaac attgggtttt gcccccaaga ccaagacctg gtagaagatc atcttctcac 960 aacactctac ctgccaacaa caccaataat attttaaatg ttggccctaa cagcaggaac 1020 agtagtaata ataataataa taataacatc atttcgaata ggaaacaagc ttccaaagaa 1080 aagaggaaaa taccaagaca tatccagaca atcgatgaaa agctaataaa cgactcgaat 1140 tacctcgcat ttttgaagtt cgatgacttg gaaaatgaaa agtttcattc ttctgcctcc 1200 tccatttcat ctccatctta ttcatctcca tctttttcaa gttatagaaa tagaaaaaaa 1260 tcagaattca tggacgatga aagctgcacc gatgtggaaa ccattgctgc tcacaacagt 1320 ctgctaacaa aaaaccatca tatagattct tcttcaaatg ttcacgcacc acccacgaaa 1380 aaatcaaagt tgaacgactt tgatttattg tccttatctt ccacatcttc atcggccact 1440 ccggtcccac agttgacaaa agatttgaac atgaacctaa attttcataa gatccctcat 1500 aaggcttcat tccctgattc tccagcagat ttctctccag cagattcagt ctcgttgatt 1560 agaaaccact ccttgcctac taatttgcaa gttaaggaca aaattgagga tttgaacgag 1620 attaaattct ttaacgattt cgagaaactt gagtttttca ataagtatgc caaagtcaac 1680 acgaataacg acgttaacga aaataatgat ctctggaatt cttacttaca gtctatggac 1740 gatacaacag gtaagaacag tggcaattac caacaagtgg acaatgacga taatatgtct 1800 ttattgaatc tgccaatttt ggaggaaacc gtatcttcag ggcaagatga taaggttgag 1860 ccagatgaag aagacatttg gaattattta ccaagttcaa gttcacaaca agaagattca 1920 tcacgtgctt tgaaaaaaaa tactaattct gagaaggcga acatccaagc aaagaacgat 1980 gaaacctatc tgtttcttca ggatcaggat gaaagcgctg attcgcatca ccatgacgag 2040 ttaggttcag aaatcacttt ggctgacaat aagttttctt atttgccccc aactctagaa 2100 gagttgatgg aagagcagga ctgtaacaat ggcagatctt ttaaaaattt catgttttcc 2160 aacgataccg gtattgacgg tagtgccggt actgatgacg actacaccaa agttctgaaa 2220 tccaaaaaaa tttctacgtc gaagtcgaac gctaaccttt atgacttaaa cgataacaac 2280 aatgatgcaa ctgccaccaa tgaacttgat caaagcagtt tcatcgacga ccttgacgaa 2340 gatgtcgatt ttttaaaggt acaagtattt tgaaataggc atgttgcaat aaaacgaaaa 2400 caactaaaaa tcacgaaaac aaaatgatat tatacaataa aaaattctta ttatgggtaa 2460 tgatagtatt cttcgcctgc ttaggcgtcc ttttccttca acaacaaaaa ttccaaaaaa 2520 aaaaagtaaa aaaacaaaac tttgattgtt ttttaatgat gttaatgatt ttttttttct 2580 ttctttatca taaaaaaaaa gttaaaatga aaaacaaata tgggtctgga aggccattat 2640 ttttttttta tttatatacc gtttctggta cttagttatt tattctcata catacactat 2700 attcaaatta cctaagagca ttttcacata tccgtttact ttcatttttt ttttttttgc 2760 ttccttttta catatcttcc gtatatcaca tcacgtttac gcgtatggtg aaacacgtca 2820 agagaaaaat gataaaatca aattttgatt tacatcaggc tccacaggac agggaaatct 2880 atctagtgag gcgataactg tagttcgatg tactcatttg aactggacaa attgaaaatt 2940 gagctgaaaa catgggagca tgatttcatt gataaaaata aaagggaacc cacaagggat 3000 gacatcaaga gcctgcggac tgttcggcag atgtataaac aatattccac actgaagaag 3060 aaacaatctt tgcaacgaca aaaagttgac actcaagagt cggttgaact cccggcacat 3120 aaaaaagacc acgacgaagt cgtagagata ggccctactc cccaagttta cggtaaggcg 3180 attagtatct ttgacatgaa tttgtcgcct ataaagccta tatacatgac attcacaaat 3240 aatattgatg ttaacaatga taactccaag acaatttcta atgaatcttc tccacgaaaa 3300 actattctcc taaaatcgtc gcctgcagga attcgatatc aagcttatcg ataccgtcga 3360 9 3081 DNA Artificial Sequence Description of Artificial Sequence DNA construct consisting of the TEF2 promoter fused to the coding region of HAP4 9 agctcaatgt ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc 60 gtaccacttc aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg 120 tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt 180 ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt 240 tttttgattt ttttctcttt cgatgacctc ccattgatat ttaagttaat aaacggtctt 300 caatttctca agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgc 360 tcattagaaa gaaagcatag caatctaatc taagttttaa ttacaaatct agaactagtg 420 gatcccccca tgaccgcaaa gacttttcta ctacaggcct ccgctagtcg ccctcgtagt 480 aaccatttta aaaatgagca taataatatt ccattggcgc ctgtaccgat cgccccaaat 540 accaaccatc ataacaatag ttcgctggaa ttcgaaaacc atggcagtaa aaagaagaag 600 aagtctagct tggtggttag aacttcaaaa cattgggttt tgcccccaag accaagacct 660 ggtagaagat catcttctca caacactcta cctgccaaca acaccaataa tattttaaat 720 gttggcccta acagcaggaa cagtagtaat aataataata ataataacat catttcgaat 780 aggaaacaag cttccaaaga aaagaggaaa ataccaagac atatccagac aatcgatgaa 840 aagctaataa acgactcgaa ttacctcgca tttttgaagt tcgatgactt ggaaaatgaa 900 aagtttcatt cttctgcctc ctccatttca tctccatctt attcatctcc atctttttca 960 agttatagaa atagaaaaaa atcagaattc atggacgatg aaagctgcac cgatgtggaa 1020 accattgctg ctcacaacag tctgctaaca aaaaaccatc atatagattc ttcttcaaat 1080 gttcacgcac cacccacgaa aaaatcaaag ttgaacgact ttgatttatt gtccttatct 1140 tccacatctt catcggccac tccggtccca cagttgacaa aagatttgaa catgaaccta 1200 aattttcata agatccctca taaggcttca ttccctgatt ctccagcaga tttctctcca 1260 gcagattcag tctcgttgat tagaaaccac tccttgccta ctaatttgca agttaaggac 1320 aaaattgagg atttgaacga gattaaattc tttaacgatt tcgagaaact tgagtttttc 1380 aataagtatg ccaaagtcaa cacgaataac gacgttaacg aaaataatga tctctggaat 1440 tcttacttac agtctatgga cgatacaaca ggtaagaaca gtggcaatta ccaacaagtg 1500 gacaatgacg ataatatgtc tttattgaat ctgccaattt tggaggaaac cgtatcttca 1560 gggcaagatg ataaggttga gccagatgaa gaagacattt ggaattattt accaagttca 1620 agttcacaac aagaagattc atcacgtgct ttgaaaaaaa atactaattc tgagaaggcg 1680 aacatccaag caaagaacga tgaaacctat ctgtttcttc aggatcagga tgaaagcgct 1740 gattcgcatc accatgacga gttaggttca gaaatcactt tggctgacaa taagttttct 1800 tatttgcccc caactctaga agagttgatg gaagagcagg actgtaacaa tggcagatct 1860 tttaaaaatt tcatgttttc caacgatacc ggtattgacg gtagtgccgg tactgatgac 1920 gactacacca aagttctgaa atccaaaaaa atttctacgt cgaagtcgaa cgctaacctt 1980 tatgacttaa acgataacaa caatgatgca actgccacca atgaacttga tcaaagcagt 2040 ttcatcgacg accttgacga agatgtcgat tttttaaagg tacaagtatt ttgaaatagg 2100 catgttgcaa taaaacgaaa acaactaaaa atcacgaaaa caaaatgata ttatacaata 2160 aaaaattctt attatgggta atgatagtat tcttcgcctg cttaggcgtc cttttccttc 2220 aacaacaaaa attccaaaaa aaaaaagtaa aaaaacaaaa ctttgattgt tttttaatga 2280 tgttaatgat tttttttttc tttctttatc ataaaaaaaa agttaaaatg aaaaacaaat 2340 atgggtctgg aaggccatta tttttttttt atttatatac cgtttctggt acttagttat 2400 ttattctcat acatacacta tattcaaatt acctaagagc attttcacat atccgtttac 2460 tttcattttt tttttttttg cttccttttt acatatcttc cgtatatcac atcacgttta 2520 cgcgtatggt gaaacacgtc aagagaaaaa tgataaaatc aaattttgat ttacatcagg 2580 ctccacagga cagggaaatc tatctagtga ggcgataact gtagttcgat gtactcattt 2640 gaactggaca aattgaaaat tgagctgaaa acatgggagc atgatttcat tgataaaaat 2700 aaaagggaac ccacaaggga tgacatcaag agcctgcgga ctgttcggca gatgtataaa 2760 caatattcca cactgaagaa gaaacaatct ttgcaacgac aaaaagttga cactcaagag 2820 tcggttgaac tcccggcaca taaaaaagac cacgacgaag tcgtagagat aggccctact 2880 ccccaagttt acggtaaggc gattagtatc tttgacatga atttgtcgcc tataaagcct 2940 atatacatga cattcacaaa taatattgat gttaacaatg ataactccaa gacaatttct 3000 aatgaatctt ctccacgaaa aactattctc ctaaaatcgt cgcctgcagg aattcgatat 3060 caagcttatc gataccgtcg a 3081 

What is claimed is:
 1. A method for preparing a yeast having multiple pathways, wherein the preferred metabolic pathway in the presence of a glucose or sugars giving glucose repression as a carbon source is limited or circumvented, comprising: transforming the yeast with a nucleic acid construct containing a gene encoding a transcriptional activator for at least one gene encoding an enzyme in one of said pathways under conditions wherein the transcription activator is over expressed causing glucose-repressed genes to derepress or circumvent the repression of one metabolic pathway which is not preferred in the presence of said carbon source and wherein the transcription activator is HAP4 or a functional equivalent or a fragment thereof wherein the molecule still has the same activity as HAP4 in transactivation.
 2. A method according to claim 1 whereby the yeast is Saccharomyces.
 3. A method according to claim 2 whereby the Saccharomyces is a Saccharomyces cerevisiae.
 4. A method according to claim 1 whereby the repressed metabolic pathway includes the respiratory or glyconegesis pathway.
 5. A method according to claim 1 whereby the pathways for metabolism of the non-preferred carbon sources are activated.
 6. A method according to claim 1 whereby the transcriptional activator is provided by introduction into the yeast of a recombinant nucleic acid encoding said activator.
 7. A method according to claim 6 whereby said recombinant nucleic acid is an expression vector.
 8. A method according to claim 6 whereby the recombinant nucleic acid is derived from the same species as the yeast.
 9. A method according to claim 1 whereby the transcriptional activator is constitutively expressed by said yeast.
 10. A method according to claim 1 whereby the transcriptional activator is expressed by the yeast upon induction.
 11. A method according to claim 10 whereby expression of said activator is induced by the presence of glucose.
 12. A method according to claim 1 whereby the yeast comprises a recombinant nucleic acid encoding a protein of interest.
 13. A method according to claim 12 whereby said recombinant nucleic acid is an expression vector.
 14. A method according to claim 12 whereby said protein of interest is a heterologous protein.
 15. A yeast obtained by the method according to claim
 1. 16. A yeast according to claim 15 having improve biomass upon culturing.
 17. A yeast according to claim 15 showing increased glucose oxidation.
 18. A yeast according to claim 15 displaying increased oxidative sugar metabolism.
 19. A yeast according to claim 15 displaying reduced production of ethanol.
 20. A yeast according to claim 15 which under anaerobic culturing conditions behaves essentially the same as the corresponding unmodified yeast. 